| Abstract No.: | B-B2064 |
| Country: | Canada |
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| Title: | THE SEPTIN 5 PROTEIN IS A MOLECULAR DETERMINANT OF CA2+ CHANNEL- SYNAPTIC VESICLE SPATIAL COUPLING AT THE DEVELOPING CALYX OF HELD SYNAPSE. |
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| Authors/Affiliations: | 1 Michael J. Fedchyshyn*; 1 Chris W. Tsang;1 William S. Trimble; 1 Lu-Yang Wang;
1 Hospital for Sick Children & University of Toronto, ON, Canada
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| Content: | Neurotransmitter release at the calyx of Held synapse employs different spatial arrangements of voltage gated Ca2+ channels (VGCCs) and synaptic vesicles (SVs) during development. In immature synapses, VGCCs are spatially distant from SVs (Micromain Modality), with release depending on the cooperative action of many loosely-coupled VGCCs to raise [Ca2+] to fusion threshold. With maturation, the coupling between VGCCs and SVs tightens where fewer tightly-coupled VGCCs are required to release SVs (Nanodomain Modality). However, molecular determinant(s) involved in this developmental switch in release modality remains unknown. Septin 5 (Sept5) forms actin filaments and has been shown to act as a molecular “brake” preventing SV fusion in secretary cells. Here we investigated whether Sept5 plays a role in subsyanptic organization in the nerve terminal. Using antibody labelling of Sept5, transmission electron microscopy (tEM), paired recordings of presynaptic Ca2+ currents (ICa) and excitatory postsynaptic currents (IEPSC), we examined morphological and functional properties of the calyx of Held synapse from immature (P8-12) and mature (P16-18) Sept5+/+ and Sept5-/- mice. We found that Sept5 was colocalized with VAMP in the presynaptic terminal of Sept5+/+ synapses, but completely absent in Sept5-/- synapses. Analyses of subsynaptic distribution patterns of SVs with tEM revealed much more docked vesicles on active zones (AZs) in Sept5-/- calyces than in Sept5+/+ calyces of immature synapses, suggesting that a deletion of Sept5 caused SVs to localize more tightly to AZs where VGCCs presumably reside. Mature synapses, with already many docked SVs, showed no difference in the SV distribution patterns. By measuring input (ICa)-output (IEPSC) relationships, we found a significant reduction in the “Ca2+ domain cooperativity” in immature Sept5-/- synapses (~3, vs. 5 in Sept5+/+), suggesting a tightened coupling between VGCCs and SVs. In parallel, Ca2+-dependent shortening in synaptic delay (SD), prominent in Sept5+/+ synapses was markedly attenuated in Sept5-/- synapses. In response to train stimuli (200Hz, 200ms), we found that SD in immature Sept5+/+ synapses shortens during facilitation but prolongs during depression in IEPSC. In Sept5-/- synapses, the shortening in SD during facilitation was eliminated, leaving prolongation in SD unaffected. These observations demonstrated that a deletion of Sept5 in immature synapses alter subsynaptic distribution of SVs near AZs, tightens functional coupling between VGCCs and SVs, and short-term changes in SD, reminiscent of mature synapses. We suggest that Sept5 is one of the key molecular determinants involved in establishing spatial arrangements of VGCCs and SVs and hence developmental transformation of release modality at the calyx of Held synapse.
Support Contributed By: OGS, BWF & CIHR
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